A 17-bp region between the −31 and −15 bp region of the mouse integrin αv gene is known to be one of the cis-acting elements for promoter activity. Experimental binding of nuclear proteins to the −31/−15 region reveals that the −27/−16 region mediates the binding. The −27/−16 region, GGCTCCTCCTCC, has a TCCTCC motif, one of the Sp1 binding motifs. An anti-Sp1 IgG and an Sp1-binding oligonucleotide interfered with the binding of nuclear proteins to the −27/−16 oligonucleotide, demonstrating that Sp1 binds to the −27/−16 region. In addition to the −27/−16 region, two other regions, −108/−89 and −64/−44, were found to bind to nuclear proteins within the −108/+1 αv promoter region. An oligonucleotide containing the Ets-binding consensus sequence of CAGGAAGT interfered with their binding, indicating that both regions have a functional Ets-binding site; which is ACGGAAGT from −106 to −99 bp and ACTTCCTC from −61 to −54 bp, as deduced from the sequence. Mutations in or deletions from any one of three cis-acting elements, the two Ets-binding sites or one Sp1-binding site, remarkably decreased the promoter activity detected in the −108/+1 region. Cotransfection of both Sp1 and Ets-1 cDNAs with the −108/+1 region into B16F10 cells increased the promoter activity 2.9-fold. These results demonstrate that Sp1 and Ets cooperate to activate the −108/+1-αv promoter region.